The glycopeptides (GP) vancomycin (Vm) and teicoplanin (Tco) are the agents of choice in treating infections caused by methicillin-resistant staphylococci which are often multiply resistant to other commonly used agents. GP resistance has been recognized among the less-pathogenic coagulase-negative staphylococcal species for many years. Of great concern has been the recent emergence of intermediate GP resistance in methicillin-resistant S. aureus isolates obtained from patients who did respond to Vm therapy in Japan and the United States. Although many biochemical correlates of GP resistance among staphylococci have been identified, the mechanism of resistance is still unclear. Moreover, techniques for identifying resistant isolates in the clinical laboratory have proved insufficient and unreliable. Therefore, random approaches to scan the genome of GP-resistant clinical isolates for determinants of GP resistance may prove to be an effective means to identify the resistance mechanism. We propose to perform several complementary random genomic scanning techniques, transposon (Tn) mutagenesis, screening of plasmid DNA libraries produced from GP-resistant clinical isolates and mRNA differential display. Tn mutagenesis of GP-resistant isolates may lead to identification of factors essential for GP resistance. Screening plasmid libraries will identify loci sufficient for resistance and mRNA differential display will lead to the identification of genes that are differentially expressed between GP susceptible and resistant strains. Putative determinants identified by these techniques will be used in complementation analyses and insertional inactivation experiments to establish their role in resistance. We have already isolated a Vm-susceptible Tn551 mutant from a laboratory-derived Vm-resistant strain, 523k. We propose to identify the locus flanking the Tn551 insertion in 523k responsible for decreasing the resistance phenotype. The GP-resistant clinical isolates will also be used directly in the Tn mutagenesis studies, library screening and mRNA differential display. These strategies could reveal the mechanism of resistance leading to identification of novel targets for antimicrobial therapy and detection of resistant isolates.